Project Summary Arsenic is the most prevalent toxin in the environment. This natural metalloid enters the biosphere from geochemical sources and, to a lesser degree, from anthropogenic sources. Human exposure to arsenic is mainly through food, water and air, and contamination of groundwater poses a worldwide health problem. Inorganic aqueous arsenic exists mainly as oxyanions of trivalent arsenite [As(III)] and pentavalent arsenate [As(V)]. As(V) is much less toxic than As(III), which is thiol reactive and binds covalently to cysteine residues in proteins. Chronic exposure to inorganic arsenic is associated with cardiovascular disease and hypertension, diabetes mellitus, neurological disorders, and various forms of cancer. It has been proposed that both direct modification of biomolecules by As(III) and reactive oxygen species (ROS) generated by arsenicals are responsible for its toxicity and carcinogenicity. Despite these health effects, As(III) is used as a highly effective treatment for certain types of cancers. Therefore, it is important to understand the cellular responses mobilized by arsenic-induced stress. Both As(V) and As(III) exposure stimulate the yeast stress-activated MAPK (SAPK) Hog1, whose activity is critically important for the cellular response to arsenic. We are interested in two general questions. First, how do diverse stressors activate a small number of SAPKs? We have found that many stressors activate yeast SAPKs by intracellular routes that interface with SAPK pathways in atypical ways, rather than signaling from the cell surface, which may influence the behavior of the SAPK. Second, how does the cell mobilize coherent, stress-specific outputs from an activated SAPK? This proposal centers on the cellular responses to arsenic exposure. We have developed evidence that both As(III) and its methylated metabolite, MAs(III), are important signaling molecules that allow cells to mobilize protective, stress-specific responses through modification of specific cysteine residues in target proteins. We refer to this as an arsenic stress signaling code. Aim1 extends our recent findings that cells respond differently to As(V) and As(III) exposure. We propose to understand the mechanistic bases of distinct regulatory events driven by these stressors. We will identify key targets of arsenic modification for the regulation of the glycerol channel Fps1 [the major port of entry for As(III)], and test the role of newly discovered arsenic modifications of proteins involved in the regulation of the oxidative stress response and replication initiation. Aim 2 is to understand how Hog1 activated by As(III) drives stress-specific outputs. This aim extends our recent finding that Hog1 itself is modified by arsenic and that this modification is important for its role in the response to As(III). Using mass spectral approaches, we will determine the Hog1 phosphorylome in response to As(III) and As(V) and establish whether Hog1 target specificity is altered by arsenylation. Aim 3 is to delineate the novel pathway by which As(V) activates Hog1 and to determine its significance for As(V) entry to cells. Completion of these aims will establish a novel paradigm centered on the regulatory nature of protein arsenylation.